In this study, neem leaves were successively extracted with petroleum ether, 95% ethanol and water and the insecticidal activities of these extracts against Oxya chinensis larvae were measured. The results showed that 95% ethanol extract gave the highest extraction yield and insecticidal activity, and it was further extracted with five different solvents. The petroleum ether extract from the 95% ethanol extract possessed the highest insecticidal activity with median lethal concentration values ranging from 14.93 to 55.66mg/mL. The gas chromatography-mass spectrometer analysis showed that the petroleum ether extract mainly composed of alkanes, olefin, esters and amide. The pathological examination revealed that the prominent lesions, including reduced regenerative cells in midgut and swelled and degenerated cylindrical cells, were observed in the 5th instar Oxya chinensis after treatment. The ultrastructural features showed that the cylindrical cells, microvilli and mitochondria were seriously damaged. These results suggested that the petroleum ether extract from neem leaves had potent insecticidal activity and could be a candidate insecticide.

Oxya chinensis (Orthoptera: Acridoidae) mainly distributed in Africa, Oceania and Asia. In China, O. chinensis is distributed throughout the whole country except Tibet and Qinghai provinces (Yin et al., 2008). O. chinensis is a hazardous pest threatening the production of rice and other gramineous plants. The traditional control method relies on chemical pesticides. However, most of them have drawbacks such as resistance, toxicity and environmental contamination (Ba et al., 2013). At present, many bio-pesticides have been applied to control locusts, such as Metarhizium anisopliae, Nosema locustae, Beauveria bassiana, nematodes, entomopoxvirus and egg parasitic wasps (Tounou et al., 2008; Zha et al., 2011; Bitsadze et al., 2013). But it had been proved that these bio-pesticides were not effective enough to control locusts. For a long time, people have been looking for active insecticidal substances from natural products.

Azadirachta indica A. Juss (syn Melia azadirachta L.) commonly known as neem, possessed biocidal activities such as insecticidal and fungicidal, and neem-based pesticides have been recognized as one of the best bio-pesticides all over the world (Montes-Molina et al., 2008; Javed et al., 2008; Anjorin et al., 2008). Azadirachtin isolated from neem was found to have biocidal activity against nearly 400 species of pests (Srinivasan et al., 2011). Neem-based pesticides with excellent insecticidal activity against locusts are labeled as ideal pesticides (Zhang et al., 2004). Extracts of neem with more than 300 known components affect the physiology and behavior of a wide range of insects, mites and nematodes (Wakil et al., 2014; Ebadollahi et al., 2013; Adebayo and Krettli, 2011; Okafor et al., 2010; Du et al., 2008; Tan and Luo, 2011; Simpson et al., 2011). Many compounds with diverse chemical structures and different modes of action are classified as botanical insecticides. In Orthoptera (such as grasshoppers, crickets, locusts), the antifeedant effect seems especially important. A number of species refuse to feed on neem-treated plants for several days, sometimes several weeks (Montes-Molina et al., 2008; Lehman et al., 2007; Sharma et al., 2008).

Currently, some compounds that isolated from neem showed insecticidal activity against O. chinensis, such as saponins and azadirachtin (Amtul and Shakoori, 2014). In this paper, our objectives were to isolating the insecticidal active fraction from extracts of neem leaves by column chromatography and studying their insecticidal activity against the O. chinensis nymph in vitro and the mechanism of action by the light microscope and transmission electron microscopic.

MATERIALS AND METHODS

Neem (Azadirachta indica A. Juss) leaves were collected from Panzhihua city in Sichuan Province of China, which was identified by the Pharmacy Laboratory of Sichuan Agricultural University.

Oxya chinensis nymph were collected from the farm of Sichuan Agricultural University, reared in breeding boxes at the condition of 25±2℃, 16:8 light duration and 70±10% relative humidity, and fed with fresh corn leaves for 2d. The 5th instar Oxya chinensis were used in the experiments.

Neem leaves were successively extracted with petroleum ether, 95% ethanol and water. Each extract was taken to dryness under vacuum at 40℃ and used for the assay of their insecticidal activity. The extraction rate (%) was calculated as follows:

Extractionrate(%)=[weightofextract(g)/weightofneemleaves(g)]×100%

The insecticidal activity was evaluated by leaf-soaking and larva-soaking methods. Three extracts were diluted to 25mg/mL with water and a small amount of Tween-80 (1%). Tested insects were dipped into three extractions for 10s respectively, and then fed with the corn leaves which were dipped by the same diluted extraction for 3s. The insecticidal activity was measured by the mortality after 24h, 48h, and 72h. Water and tween-80 was used as negative control, and the whole procedure was repeated thrice. The mortality was calculated by the formulas below:

Where “n” is the number of nymph, “T” is the treated group and “C” is the control group.

The 95% ethanol extract suspended in water was successively extracted with petroleum ether, chloroform, ethyl acetate and n-butanol, respectively. Then, the petroleum ether extract was fractionated by column chromatography over silica gel G (100~200 mesh) eluted with a-hexane/ethyl acetate/acetone (8.5:1:0.5, v/v/v) mixture to give three fractions （F1-F3）(Du et al., 2009). The five extractions and three fractions (F1-F3) were diluted to 50, 25, 12.5, 6.25 and 3.12 mg/mL with water and Tween-80, respectively, and their insecticidal activities were measured as described above.

The main components of F1 were analyzed by GC-MS (Agilent 6890-5973N). The Agilent HP-5 column was used and the oven temperature program was as follows: 100℃ for 3 min; 100℃ to 120℃, 5℃/min; 120℃ to 280℃, 10℃/min; 280℃ for 10min. The injector and detector temperatures were 270℃ and 280℃, respectively. The Full-scan molecular weight ranged from 45 to 750.

The midguts tissue of the nymph from post-treatment by the petroleum ether extract after 24h, 48h and 72h were subjected to histological examination. They were pressed in a fixation medium of 10% solution of buffered formalin (pH 7.4) and enclose in paraffin-intended subsequent histopathological examination. A 5μm section of each organ was stained with hematoxylin and eosin. Each section was examined under an optical microscope.

The transmission electron microscopy assay was conducted using previously established methods (Fichi et al., 2007). The midguts tissue of the nymph was cut off after post-treatment by the petroleum ether extract for 24h, 48h and 72h and fixed by glutaraldehyde precooling (2.5%, pH 7.2). The tissue was then further fixed with 1.0% osmic acid followed by dehydration with an acetone gradient prior to embedding. Ultra-thin longitudinal sections were cut and stained with uranyl acetate. The sections were then observed using a transmission electron microscope (JEM-1010, Joel, Japan) (Hu et al., 2015).

All results were expressed as a mean±standard deviation (S.D.) for the indicated number of experiments. Groups were compared using variance analysis and the Duncan’s multiple comparison test (DMRT) by using DPS data processing system. A value of P＜0. 05 was considered significant.

RESULTS

The extraction rates of the crude extracts of neem leaves were shown in Table 1. The extraction rates of water, 95% ethanol and petroleum ether extract were 46.80%, 25.93% and 2.20%, respectively. The insecticidal activities of the crude extracts of neem leaves against 5th instar Oxya chinensis was shown in Table 2. The insecticidal activities of the three extracts were 0, 10% and 10%, respectively in 24h, 0, 16.67% and 20%, respectively in 48h and 0, 26.67% and 33.33%, respectively in 72h. The petroleum ether extract was found to have the highest insecticidal activity, and the water extract had no insecticidal activity. The 95% ethanol extract had a little lower insecticidal activity, but much higher extraction rate than the petroleum ether extract. Thus, the 95% ethanol extract was further studied.

The difference between data with the different small letter within a column is significant (P＜0.05).

The 95% ethanol extract are further purified with petroleum ether, chloroform, ethyl acetate and n-butanol in sequence extracted method, respectively. Mortalities of the Oxya chinensis nymph treated with the five extracts was shown in Table 3. At a concentration of 25mg/mL, the mortalities of the petroleum ether extract were 23.33%, 73.33%, 80%; and the mortalities of the chloroform extract were 23.33%, 30%, 43.33%, in 24h, 48h, and 72h, respectively. The ethyl acetate, n-butanol and water extracts were found to have no significant insecticidal activity in all portions. These results indicated that the petroleum ether extract showed the highest insecticidal activity than other extracts in different time. Therefore, it was necessary to conduct a further study on the petroleum ether extract.

The difference between data with the different small letter within a column is significant (P＜0.05).

The toxicity results of the petroleum ether extract are shown in Table 4, the median lethal concentration (LC50) value of the petroleum ether extract to 5th instar Oxya chinensis are 55.66mg/mL, 20.07mg/mL and 14.93mg/mL in 24h, 48h and 72h, respectively. These results indicated that the insecticidal activity of petroleum ether extract showed the relation of time-and concentration-dependent.

The difference between data with the different small letter within a column is significant (P＜0.05).

Three fractions (F1-F3) obtained from the petroleum ether extract by column chromatography were tested for insecticidal activities against 5th instar Oxya chinensis. The results were shown in Table 5 to 7. F1 showed the highest insecticidal activity. It has also shown that the three fractions (F1-F3) displayed insecticidal activity against 5th instar Oxya chinensis in all test time and concentrations.

Under light microscope, the tissue showed normal structure in the control group (Figure 1A). In the experiment group, after 24h treatment, muscle layer of midgut tissue was complete; layer of intestinal wall cells thickened and part of intestinal wall cells fell off; many nuclei of regenerative cells became pyknotic; the regenerative cells were filled with red mesh-like material; cylindrical cells swelled and part of them fell off (Figure 1B). After 48h of treatment, muscle layer of midgut tissue was complete; large area of the layer of intestinal wall cells fell off; microvilli disappeared absolutely; regenerative cells of midget were filled with red mesh-like material and nuclei became smaller; cylindrical cells swelled and lots of them occurred fatty degeneration (Figure 1C). After 72h treatment, layer of intestinal wall cells almost fell off; leaving only the circular muscle layer which became thinner and stained lighter; circular muscle layer of midget occurred vacuolar degeneration (Figure 1D).

Figure 1 Histopathological changes in the midgut of the Oxya chinensis larvae treated with the petroleum ether extract for different times. A: Control group (H.E. ×400), the tissue showed normal structure. B: After 24h treatment (H.E. ×400), layer of intestinal wall cells thickened and part of intestinal wall cells fell off; many nuclei of regenerative cells became pyknotic; the regenerative cells were filled with red mesh-like material; cylindrical cells swelled and part of them fell off. C :After 48h treatment ( H.E. ×400), large area of the layer of intestinal wall cells fell off; microvilli disappeared absolutely; regenerative cells of midget were filled with red mesh-like material and nuclei became smaller; cylindrical cells swelled and lots of them occurred fatty degeneration. D:After 72h treatment (H.E. ×200), layer of intestinal wall cells almost fell off; leaving only the circular muscle layer which became thinner and stained lighter; circular muscle layer of midget occurred vacuolar degeneration.

Under electron microscopic transmission, in the control group, rough endoplasmic reticulums were orderly with a lot of ribosomes (Figure 2A). After 24 h of treatment, rough endo-plasmic reticulum decreased in the cytoplasm and the ribosomes began to fall (Figure 2B). After 48 h of treatment, rough endoplasmic reticulums were disorganized and ribosomes fell off (Figure 2C). After72 h treatment, rough endoplasmic reticulums expanded extremely, fractured and vacuolated (Figure 2D).

Figure 2 Effects of the petroleum ether extract on the midgut of Oxya chinensis larvae. Control group (A): rough endoplasmic reticulums were orderly with a lot of ribosomes (×15000). Experiment group: after 24h treatment (B), rough endo-plasmic reticulum decreased in the cytoplasm and the ribosomes began to fall (×12000); after 48h treatment(C), rough endoplasmic reticulums were disorganized, expanded, and ribosomes fell off (×17000); after 72h treatment(D), rough endoplasmic reticulums expanded extremely, fractured and vacuolated (×10000).

In the control group, the microvilli of midgut of the Oxya chinensis nymph were orderly (Figure 3A). After 24 h of treatment, microvilli arranged neatly; the pathologic changes were not obvious (Figure 3B). After 48 h of treatment, microvilli fractured and fell off (Figure 3C). After 72 h of treatment, microvilli disappeared and a lot of vacuoles appeared (Figure 3D).

Figure 3 Effects of the petroleum ether extract on the midgut of Oxya chinensis larvae. Control group (A), the microvilli of midgut of the Oxya chinensis larvae was orderly (×6000). Experiment group: after 24h treatment (B), microvilli arranged neatly, the pathologic changes were not obvious (×12000); after 48h of treatment (C), microvilli fractured and fell off (×15000); after 72h of treatment (D), microvilli disappeared and a lot of vacuoles appeared (×10000).

In the control group, the mitochondria of the dermis cells were distributed evenly; crest clarity was regularly arranged in the plate layer; the surrounding membrane was clearly observed (Figure 4A). After 24 h of treatment, mitochondria swelled in different degrees; cristae shortened and decreased (Figure 4B). After 48 h of treatment, mitochondria swelled obviously; a matrix of mitochondria occurred at electronic clear area; cristae almost disappeared (Figure 4C). After 72 h of treatment, mitochondria swelled extremely; many vacuoles occurred and broke; myelin figures were observed (Figure 4D).

Neem belonging to the Meliaceae family has been investigated intensively for its potential as a bio-insecticide (Verma et al., 2007; Farooq et al., 2011). The insecticidal activity of neem generally attributes to azadirachtin which is a well-known potent insecticide (Zheng et al., 2011; Kumar et al., 2010). 0.3% azadirachtin EC (150mL/hm2 and 210 hm2) was sprayed in the grassland to investigate the control effect on grasshoppers. The results showed that the effects of controlling Calliptamus abbreviate ikovnnikov and Oedaleus decorus asiaticas after 3d were 90.6% and 92.5% (Yang et al., 2006). In this study, the results showed that the petroleum ether extract from neem leaves had obvious stomach toxicity against O. chinensis nymph from the toxic symptoms and displayed insecticidal activity against 5th instar O. chinensis in all test time and concentrations in a time-and concentration-dependent manner. The present study may encourage further researches on using simple and inexpensive application methods for controlling grasshoppers.

The main components of the petroleum ether extract of neem leaves were 6 alkanes, 1 olefin, 2 esters and 1 amide. However, the phytochemical analysis did not reveal the presence of Azadirachtin, which has been considered as one of the main active compounds of neem. These results suggested that Azadirachtin was excluded from the petroleum ether extract during our separation process and other compounds from the extract of neem leaves also possessed potent insecticidal activity and could be used as insecticide. In the previous study, we found that octadecanoic acid-tetrahydrofuran-3, 4-diyl ester (ODA-THF) exhibited acaricidal activity against Sarcoptes scabiei var. cuniculi nymph (Du et al., 2009), which will be subject of more studies in further experiments.

The petroleum ether extracts damaged the pivotal organs of the digestive system which was also one of the causes of death. The important reason was the peritrophic membranes disappearance and the midgut cells destruction. The peritrophic membranes are the first obstacle through the foods pass through the digestive tract wall and are an important target for many pesticides (Hegedus et al., 2009). The observed histopathological effects of the petroleum ether extract on the midgut tissue of O. chinensis nymph were similar to the results on Mythimna separate and Plutella xylostella (Lü et al., 2010). It stated that the midgut of O. chinensis was affected after treatment with the petroleum ether extract and the midgut membrane system was damaged seriously. All the above observations showed that damaging the normal structure of midgut and endomembrane may be one of the primary mechanisms.

Transmission electron microscopy results showed that the autolysis of midgut cells was the main cause of the changes. After 48h of treatment, the results showed that midgut cells began to change. The microvilli showed fractures, fell off, and disappeared; partial vacuolation formed. The lysosome increased and organelle started autolysis. Based on the above results, it could be concluded that one of the main reasons was that the midgut cells fell off. But the protease activity of midgut, such as acid phosphatase, needs to be further studied to confirm the results.

ACKNOWLEDGEMENT

This study was supported by the International cooperation projects of Sichuan Province (2014HH0058, 2013HH0042), the Sichuan Youth Science and Technology Innovation Research Team for waterfowl disease prevention and control (2013TD0015) and the National Natural Science Foundation of China (Grant No.31372477).